| The apple is the world fruit,and most countries in the world rank it with the main consuming fruits. Because polyploidy fruit trees generally have the characteristics of vigorous growing,big fruit size, fewer seeds or seedless,high production,strong adaptability and resistance,and could keep excellent characteristics with asexual propagation,polyploidy breeding is considered as the importance approach of create new apple cultivars.This study obtained apple novel polyploidy germplasm with the mature embryos of 'Golden Delicious','Gala','Fuji' and the in vitro leaf of 'Yan 1 Gala','Hanfu' as materials by colchicine induction and polypolid hybridization.Besides on that,we analyzed the difference between apple tetraploid and diploid on molecule level,which will lay a foundation for the work on elucidating the evolution mechanism of apple polyploidy.The main results are as follows:1.The system of induction of tetraploid from apple mature embryos in vitro was successfully established.The induction rates was 10.6%when the mature embryos of 'Golden Delicious' apple were treated with 0.5%colchicines and 1%DMSO for 48h and then cultured on MS medium supplemented with TDZ 1.0mg·L-1,IBA 0.5 mg·L-1.2.In vitro tetraploid induction of 'Hanfu' and 'Yan 1 gala' apple were studied by treating leaf with colchicines solution.The inducing percentage was from 5.3%to 22.2%when the leavess of 'Hanfu' were treated in liquid medium(MS+TDZ 1.0 mg·L-1+NAA 0.5 mg·L-1+LH 200 mg·L-1) supplemented with colchicines 15 mg·L-1,30 mg·L-1,60 mg·L-1 or 120 mg·L-1 for five days at dark.And tetraploid plantlets were also obtained from 'Hanfu' and 'Yan 1 gala' when the leaves were treated on solid medium supplemented with colchicines for five days at dark.The induction rates were 20.0%and 15.0% for 'Hanfu' and 'Yan 1 gala' respectively.3.The leaves of ploidy chimeras were inoculate to culture medium which contained MS+TDZ 1.0 mg·L-1+IBA 0.5 mg·L-1+sucrose30 g·L-1+ Agar 30g·L-1 for adventitious shoots regeneration.The homogeneity mutants could be separated form the 'Golden delicious','Gala' and 'Fuji' ploidy chimeras.4.Comparing the three schemes for obtaining apple triploid plants:(1) hybridization between diploid and tetraploid plant;(2) crossing between diploid and triploid plant;(3) selecting triploid plants from triploid seedling progenies.Result show that the best methed was the hybridization between diploid and tetraploid plant,as the percentage of triploid plants obviously higher than that using other schemes.5.Apple polyploid plants were vigorous with strong shoots and short internodes,and its leaves were big,round,coarse,and thick with more teeth,.Stomas of triploid seedling progenies were bigger than that of diploid and aneuploid plant,but the density of stomas was less than that in diploid and aneuploid plant. Among the polyploid seedling progenies of apple,different ploidy plants had marked discrimination in plant shape.Based on plant shape and index of leaf shape,polyploid(tetraploid and triploid) plants could easily be distinguished from the diploid plants and the aneuploid plants. 6.The chlorophyll a,chlorophyll b,the total amount of chlorophyll and carotenoid of diploid apple cultivars 'Fuji' and 'Yan 1 Gala' were below their autotetraploid 'Tensei' and 'tetraploid Yan 1 Gala', respectively.However,chlorophyll a/b ratio have not showed consistent changes.The chlorophyll fluorescence parameters changes in the law of diploid varieties 'Fuji' and 'Yan 1 Gala' and their respective autotetraploid species were measured and the results showed that FO averages of diploid varieties 'Fuji' and 'Yan 1 Gala' were 214.6 and 208.0,which are higher than the 'Tensei' and 'tetraploid Yan 1 Gala'-9.4 and 8.3.The 'PI' on diploid varieties Fuji and 'Yan I Gala' were lower than their autotetraploid 'Tensei' and 'tetraploid Yan 1 Gala' respecitively.7.The diploidy and tetraploidy pollens of apples were observed by electron microscope,the pollens have three longthways channels,most of their profiles were oblong and polars were triangle.The pollen surfaces have longways striations and perforations.The pollen quantity of tetraploids were less than that of diploids,and nonablong pollens were only few.P/E value of tetraploids was 1.8,and that of diploids was 2.0,which have significant difference.8.The 'Fuji','Yan 1 Gala' and 'Hanfu' of diploid and autotetraploid were analyzed with ISSR markers.A total of 65 bands,ranged from 180 to 2500 bp,were amplified with eight primers. Twenty-four bands were polymorphic,accounting for 36.9%,and each primer can amplify from 0 to 5 polymorphic bands,with 3 bands at average.Three groups of apples('Fuji' and 'Tensei','Hanfu' and 'tetraploid Hanfu','Yan 1 Gala' and 'tetraploid Yan 1 Gala') have difference,but the genetic similarity coefficients between diploidys and its autotetraploidys of 'Fuji','Yan 1 Gala' and 'Hanfu' all were 1,and they could not distinguish each other.9.The content of cytosine and 5-methyl cytosine in hydrolysis DNA samples extracted from leaves of the diploid of apple and its autotetraploid was identified used HPLC.The 'Tensei' had low DNA methylation which was 29.33%and 'Fuji' was 35.34%.The methylation degree of 'Hanfu' diploid and tetraploid were 14.37%and 14.49%respectively,with no significant difference.10.Two hundred and fifty-one homogeneity tetraploid trees were induced from embryos of apple cultivars 'Golden Delicious','Gala' and 'Fuji'.Twenty and thirteen tetraploid plants were induced from leaves of 'Hanfu' and 'Yan Gala 1' respectively.Forty-six triploid tress and 5 teraploid trees were obtained from the offsprings of teraploid crossing diploid,the offsprings of triploid crossing diploid,and the self-progenies of triploid.These polyploidy germplasms laid the important foundation for breeding polyploid apple cultivars.
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